Bibliographic details of the publications referred to in this specification are also collected at the end of the description.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
It is apparent that DNA methylation and other epigenetic modifications play a role in the regulation of gene expression in higher organisms. The importance of epigenetic modification has been highlighted by its involvement in several human diseases. Methylation, for example, of cytosine at the 5′ position is the only known methylation modification of genomic DNA. In particular, methylation of CpG islands within regulatory regions of the genome appears to be highly tissue specific. Methylation of cytosines distal to the islands is also important. These regions are called “shores” or “island shores” (Irizarry et al., Nature Genetics 41(2):178-186, 2009). Epigenetic modifications include histone modification, changes in acetylation, methylation, obiquitylation, phosphorylation, sumoylation, activation or deactivation, chromatin altered transcription factor levels and the like.
Another genetic condition which can affect gene expression arises from expansion or increase in the number of repeats in a specific tandem repeat array. Such nucleotide expansion can result in repeat expansion disease conditions. A critical threshold of repeat expansion determines the level of pathologenicity (Orr and Zoghbi, Ann Rev Neurosci 30:575-621, 2007). Many diseases arise from expansion of a repeat located in an open reading frame resulting in a protein with a long polyQ2 tract that is toxic to neurons (Orr and Zoghbi, 2007 supra). Other expansion disease conditions such as Fragile X syndrome (FXS), Fragile XE mental retardation (FRAXE), Fragile X-associated primary ovarian insufficiency (FXPOI), Fragile type, folic acid type, rare 12 (FRA12A), mental retardation (MR), Friedrich's ataxia (FRDA) and myotonic dystrophy (DM), arise from altered transcription of the repeats which are not translated.
A particular type of expansion disorder is referred to as a trinucleotide repeat disorder (also known as trinucleotide repeat expansion disorder, triplet repeat expansion disorder and codon reiteration disorder) and results from trinucleotide repeats in certain genetic loci. An example occurs in the Fragile X Mental Retardation genetic locus (“FMR genetic locus”).
The FMR genetic locus includes the FMR1 gene which is composed of 17 exons, spanning 38 Kb, and encodes Fragile X Mental Retardation Protein (FMRP), essential for normal neurodevelopment (Verkerk et al., Cell 65(5):905-914, 1991; Terracciano et al., Am J Med Genet C Semin Med Genet 137C(1):32-37, 2005). A CGG repeat segment is located within the 5′ untranslated region (UTR) of the gene. Its normal range is <40 repeats. When expanded, these repeats have been implicated in a number of pathologies, including the Fragile X syndrome (FXS), Fragile X-associated Tremor Ataxia Syndrome (FXTAS) and Fragile X-associated primary ovarian insufficiency (FXPOI; formerly referred to as Premature Ovarian Failure [POF]). FXS is neurodevelopmental in nature with a frequency of 1/1400 males and 1/8000 females, associated with a Fragile site at the Xq27.3 locus (Jin and Warren, Hum. Mol. Genet 9(6):901-908, 2000).
This syndrome is caused by a CGG expansion to “full mutation” (FM) which comprises >200 repeats, leading to a gross deficit of FMRP and subsequent synaptic abnormalities (Pieretti et al., Cell 66(4):817-822, 1991; Irwin et al., Cereb Cortex 10(10):1038-1044, 2000). The FXS clinical phenotype ranges from learning disabilities to severe mental retardation and can be accompanied by a variety of physical and behavioral characteristics. FXTAS is prevalent in ˜30% of premutation individuals (PM), comprising 55 to 199 repeats (Nolin et al., Am J Hum Genet 72(2):454-464, 2003) and is a progressive neurodegenerative late-onset disorder with a frequency of 1/3000 males in the general population (Jacquemont et al., Am J Ment Retard 109(2):154-164, 2004), manifesting as tremor, imbalance and distinct MRI and histological changes (Hagerman et al., Neurology 57(1):127-130, 2001; Jacquemont et al., J Med Genet 42(2):e14, 2005; Loesch et al., Clin Genet 67(5):412-417, 2005). It is often associated with ‘toxicity’ of elevated FMR1 mRNA, which has been linked to the intranuclear inclusions and cell death observed during neurodegeneration (Jin et al., Neuron 39(5):739-747, 2003).
FXTAS can occur in females carrying PM, but with much lower frequency as can be expected from X-linked inheritance. The intermediate or Gray Zone (GZ) alleles comprising 41 to 54 repeats (Bodega et al., Hum Reprod 21(4):952-957, 2006) are the most common form of the expansion, 1 in 30 males and 1 in 15 females. As with PM alleles, increased levels of FMR1 mRNA have been reported in the GZ individuals, proportional to the size of CGG expansion (Kenneson et al., Hum Mol Genet 10(14):14491454, 2001; Mitchell et al., Clin Genet 67(1):38-46, 2005; Loesch et al., J Med Genet 44(3):200-204, 2007). Female carriers of both PM and GZ allelic types have an increased risk of developing POF (Allingham-Hawkins et al., Am J Med Genet 83(4):322-325, 1999; Sullivan et al., Hum Reprod 20(2):402-412, 2005) which has incidence of approximately 1% in the general population, and often unknown etiology (Coulam, Fertil Steril 38(6):645-655, 1982).
Expansion related abnormalities in FMR1 are involved in pathologies with a wide spectrum of patho-mechanisms all pointing to involvement of multiple factors at the Xq27.3 locus in addition to FMR1. A number of antisense transcripts have been described embedded within the FMR1 sequence, ASFMR1 (Ladd et al., Hum Mol Genet 16(24):3174-3187, 2007) and FMR4 (Khalil et al., PLoS ONE 3(1):e1486, 2008). The ASFMR1 and FMR4 transcripts have been suggested to share the bi-directional promoter with FMR1, which is heavily regulated by the state of the surrounding chromatin environment (Pietrobono et al., Nucleic Acids Res 30(14):3278-3285, 2002; Chiurazzi et al., Hum Mol Genet 7(1):109113, 1998).
Transcription of ASFMR1 is also regulated by another promoter located in the exon 2 of FMR1, with the resulting transcript spanning the CGG repeat in the antisense direction (Ladd et al., 2007, supra), and an open reading frame (ORF) with the CGG encoding a polyproline peptide (Ladd et al., 2007, supra). FMR4, however, is a long non-coding RNA, involved in regulation of apoptosis (Khalil et al., 2008, supra).
The length of the CGG repeat has been reported to effect transcription of all three genes FMR1, FMR4 and ASFMR1 (Ladd et al., 2007, supra; Khalil et al., 2008, supra). However, although it is well documented that FMR1 transcription is promoter methylation dependent, linked to the CGG expansion size, the relationship between FMR4 and ASFMR1 transcription and methylation remains elusive.
One of the current problems is in the diagnosis of subjects with FM in the FMR genetic locus. Diagnostic assays targeting only the CGG expansion have hitherto been inconclusive. Therefore, currently Southern DNA analysis, which is expensive and time consuming, is used as a gold standard assay for diagnosis in many laboratories.
Despite the availability of a range of methylation and nucleotide expansion assays (see, for example, Rein et al., Nucleic Acids Res. 26:2255, 1998 in relation to methylation assays), selection of regions to amplify and screen is an important aspect of determining an epigenetic profile characteristic of a disease condition. There is a need to identify crucial regions which are associated with epigenetic change linked to a pathological condition to assay and/or therapeutically target.